Methods for modeling infectious disease and chemosensitivity in cultured cells and tissues

ABSTRACT

The present invention provides methods for utilizing a form of optimized suspension culture to examine the infectivity of pathogenic organisms and agents in human cells and tissues. Also provided are methods using a rotating wall vessel to predict chemosensitivity of cells and tissues to toxins and chemotherapeutic agents. These culture conditions potentiate spatial colocalization and three-dimensional assembly of individual cells into large aggregates which more closely resemble the in vivo tissue equivalent. In this environment, dissociated cells can assemble and differentiate into macroscopic tissue aggregates several millimeters in size. These culture conditions allow for better cellular differentiation and formation of three-dimensional cellular aggregates, more efficient cell-to-cell interactions, the in in vivo-like exchange of growth factors and greater molecular scaffolding facilitating mechanical stability for cells. The suspension culture system offers a new approach for studying microbial infectivity from the perspective of the host-pathogen interaction and also for analyzing chemosensitivity to toxins and chemotherapeutic agents.

CROSS REFERENCES TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 60/282,007, filed Apr. 6, 2001, which is fully incorporated byreference herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

The invention was made in part with United States Government supportawarded by the Department of Veterans Affairs and National Aeronauticsand Space Administration. The U.S. Government has certain rights in theinvention.

BACKGROUND OF THE INVENTION

1. Technical Field of the Invention

The present invention relates generally to the fields of microbiology,infectious diseases and drug toxicity. More specifically, the presentinvention relates to methods of modeling infectious disease andchemosensitivity with three-dimensional suspension of mammalian cellsusing a bioreactor.

2. Description of the Prior Art

An essential feature of the pathogenicity of microorganisms is theirability to infect or colonize host epithelial cells and cells in othertissues. The terms “infect” and “colonize” are used interchangeablyherein, and encompass adherence to cells, invasion, survival within, anddamage or destruction of the cells. Initial sites of bacterial infectioninclude the epithelia of the intestine, kidney, lung and bladder. Thereis a need to investigate how bacterial and other pathogens interact withthe epithelium to initiate disease, which can be through the use of bothin vitro and in vivo models of infection.

Some types of tissue culture models of infectivity are not the bestmodels of the conditions faced in vivo by pathogens. Conclusions drawnabout the interaction between pathogens and the host epithelium usingcultured epithelial cells may be limited due to the dedifferentiation ofthese cells during conventional cell culture. Many of the physiologicaldifferences between cultured cells and their in vivo counterparts arebelieved to be the result of the dissociation of cells from their nativethree-dimensional geometry in vivo to their propagation on atwo-dimensional substrate in vitro. Likewise, many of thecharacteristics of animal models fail to mimic the human disease, andanimal models present a complex system in which many variables cannot becontrolled. Accordingly, since certain in vitro nor in vivo assays donot replicate the complex environment encountered by pathogens duringthe natural course of human infection, the information gained from thesestudies may be limited.

An essential feature of the pathogenicity of Salmonella is its abilityto invade host intestinal epithelial cells. While important advanceshave been made toward understanding how Salmonella interacts with theintestinal epithelium to initiate disease, through the use of both invitro and in vivo models of infection, numerous questions have beenderived from these studies which remain to be answered. In particular,it is well-documented that important differences exist between thepathogenesis of serovar Typhimurium in human infections and inwidely-used cell culture and animal models. Specifically, tissue culturemodels of infectivity are not exact models of the conditions faced invivo by Salmonella. Conclusions drawn about the interaction betweenSalmonella and the host intestinal epithelium using cultured enterocytesare limited due to the dedifferentiation of these cells duringconventional cell culture. Many of the physiological differences betweencultured cells and their in vivo counterparts are believed to be theresult of the dissociation of cells from their native three-dimensionalgeometry in vivo to their propagation on a two-dimensional substrate in:vitro. Likewise, many characteristics of animal models fail to mimic thehuman disease, and animal models present a complex system in which manyvariables cannot be controlled. Accordingly, since neither in vitro norill vivo assays faithfully replicate the complex environment encounteredby Salmonella during the natural course of human infection, theinformation gained from these studies is limited. A high fidelityenteric cell culture model can provide new insights into studies ofSalmonella infectivity by bridging the gap between the inherentlimitations of cultured mammalian cells and intact animals.

The response of cells to various drugs is an area of great interest tothe medical field. Currently, many model systems exist for testing thechemosensitivity of cells to various chemical compounds in order toidentify new pharmaceutical agents for the treatment of cancer and otherdiseases. Conventional cell culturing techniques do not alwaysaccurately simulate conditions ill vivo favorable for the growth of sometissues. Hence, some biological properties affecting tissue response todrugs are not apparent with standard cell culturing methods.

U.S. Pat. No. 6,117,674, which is fully incorporated by referenceherein, discloses an invention relating to the propagation of a pathogenselected from the group consisting of viruses, bacteria, protozoans,parasites and fungi, by inoculating a three dimensional tissue massculture at microgravity conditions in fluid culture media in amicrogravity vessel with the pathogen. Specifically, the microgravityconditions are simulated in unit gravity by a horizontal rotating wallvessel (RWV). Growth conditions in the RWV allow for better cellulardifferentiation and formation of three-dimensional cellular aggregates,more efficient cell-to-cell interactions, the ill vivo-like exchange ofgrowth factors and greater molecular scaffolding facilitating mechanicalstability for cells. The RWV bioreactors offer a revolutionary approachnot previously applied for studying microbial infectivity from theperspective of the host-pathogen interaction and also for analyzingchemosensitivity to toxins and chemotherapeutic agents, likeantibiotics.

SUMMARY OF THE INVENTION

The present invention pertains to the use of a low shear stresssuspension cell culture system to study the infectivity of pathogenicorganisms and agents in human cells and tissues. The cell cultureconditions potentiate spatial co-localization and three-dimensionalassembly of individual cells into large aggregates, which more closelyresemble the in vivo tissue equivalent. In this environment, dissociatedcells can assemble and differentiate into macroscopic tissue aggregatesseveral millimeters in size, which are largely devoid of necrotic cores.The present invention also provides methods for predicting thechemosensitivity of cells and tissues to toxins and chemotherapeuticagents. Use of the present invention to generate 3-D aggregates thatclosely resemble the “in vivo” tissue equivalent, from a variety of celltypes has wide applications in the modeling of infectious diseases.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofthe invention presented herein.

FIG. 1 shows a morphological comparison of paraffin-sectioned normalhuman small intestine (A), paraffin-embedded Int-407 (intestinalepithelial cell) monolayer cultures (B), and paraffin-sectioned Int-4073-D aggregates (C) stained with hematoxylin-eosin. The three-dimensionalorganization is observed in a cross-section of an Int-407 3-D-construct(Panel C, arrow), morphologically reminiscent of the intestinal villiseen in normal small intestine (Panel A). In contrast, note the completelack of organization observed when Int-407 cells are cultured asstandard monolayers (Panel B). A sectioned micro-carrier bead can beseen in Panel C (*). All micrographs were obtained at 200×magnification.

FIG. 2 shows the immunohistochemical analysis of 3-D Int-407 aggregatesas compared to Int-407 monolayers. This figure demonstrates positivestaining in 3-D Int-407 tissue aggregates for collagen type II,fibronectin, sialyl Lewis A, and villin (B, D, F, and H, respectively)(100×), and the absence of staining for cytokeratin 18 (J) as comparedto Int-407 monolayers stained for the same markers (A, C, E, G, and I,respectively) (100×). Antibody negative controls for 3-D tissueaggregates and monolayers (K and L, respectively), (100×), demonstratetotal lack of staining. For reference, circular and oval lumenal areasseen in the photos are cross sections of microcarrier spheres aroundwhich cellular material is growing.

FIG. 3 shows the periodic acid-Schiff (PAS) Staining of paraffinsections of Int-407 3-D constructs. The figure shows the presence ofisolated PAS-positive cells (large arrow-heads) surrounded byPAS-negative cells. Cytodex beads (*) and some discrete areas ofextracellular matrix (small arrows) also exhibit a positive reactionwith the PAS stain. The presence of a differential PAS staining patternin paraffin sections of 3D constructs suggests the presence of amucous-producing sub-population of cells within the construct.

FIG. 4 shows confocal images of antibody stained Int-407 monolayers and3-D constructs. Confocal images of Int-407 cells grown as monolayers (B,D, F, H, J, and L) and 3-D aggregates (A, C, E, G, I, and K) stainedwith monoclonal antibodies against epithelial specific antigen (ESA)(A-B), cadherin (C-D), cytokeratin 19 (E-F, note the 150 μm beads aredelineated by a dotted outline in G), laminin (I-J), and collagen typeIV (K-L).

FIG. 5 shows transmission electron micrographs (TEM) of 3-D Int-407aggregates as compared to Int-407 monolayers. This figure demonstratesTEM images of Int-407 monolayers (A-B) and 3-D Int-407 cells (C-F). Themicrovilli observed in the monolayers (A) are poorly developed andsparse in number compared to the well-developed and abundant microvilliin the 3-D aggregates (L) (solid arrows). In addition, the monolayersshow high levels of cellular granularity (Panel B.*) indicative ofpoorly differentiated cells. Moreover, the monolayers (A-B) demonstrateinferior tight junction formation as compared to the 3-D aggregates(D-E) (concave arrows). Panel E shows the formation of tight junctionswhich nearly run the length of the margin between two 3-D cells (concavearrows). Note the presence of numerous, well-formed vacuolar-likestructures in the 3-D aggregates (D) as compared to their poorlydeveloped counterparts in the monolayers (B) (arrow heads). In Panel F,two cytodex microcarrier beads (b) can be seen which are partiallycovered with 3-D cells. The microvilli display an apical orientationtoward the luminal (L) side.

FIG. 6 shows transmission electron micrographs of desmosome formation inthe 3-D Int-407 aggregates (B) compared to Int-407 monolayers (A). Thedesmosomes in the monolayer are poorly formed and incomplete.

FIG. 7 shows scanning electron micrographs (SEM) of Salmonella withInt-407 monolayers. Electron micrographs of uninfected and infectedInt-407 monolayers are at 2,000× magnification. Uninfected monolayercontrol (A); monolayer at 10 minutes post-infection, (at an MOI of10:1), showing dramatic surface alterations, including extensiveformation of membrane blebs and several surface structures resemblingruffles (B); by 1 hour post-infection, the Int-407 cell surface appearsswollen and denuded with membrane blebs (C); at 2 hours post-infection,Int-407 cells exhibit loss of structural integrity with numerousmembrane alterations including blebs and the formation of pathologicallesions, as well as some surface bound bacteria (D)).

FIG. 8 shows scanning electron micrographs of uninfected and infected3-D Int-407 aggregates. Electron micrographs of uninfected and infected3-D Int-407 aggregates in the HARV are at 2,000× magnification.Uninfected 3-D Int-407 control showing dense masses of cells withextracellular secretions (A); 3-D Int-407 aggregates at 10 minutespost-infection (at an MOI of 10:1), showing minor irritation on theInt-407 apical cell membrane (B); by 1 hour post-infection, numeroussurface depressions and/or membrane blebs are apparent on the 3-DInt-407 cells, with depressed regions devoid of mucous-like secretions(C); at 2 hours post-infection, the surface of the 3-D Int-407 cells isquite irregular and associated with what appear to be numerous surfacelesions (D). Few surface bound bacteria were observed at any of theabove postinfection times.

FIG. 9 shows cytokine expression of Int-407 monolayers and 3-D tissueaggregates following Salmonella infection. Total RNA was isolated fromInt-407 monolayers and 3-D tissue aggregates prior to infection withSalmonella and at 1 h and 2 h after infection. Five jig total RNA fromeach sample was hybridized individually with radiolabeled riboprobesspecific for TNFα (Panel A) or IL-6 (Panel B) and subjected to RNAseprotection. Phosphorimager technology was used to quantify the levels oftranscription of each cytokine and the housekeeping gene, GAPDH. Levelsof TNFα and IL-6 transcripts were normalized using GAPDH transcriptionlevels of the same RNA sample and the levels are displayed as arbitraryunits.

FIG. 10 shows that cell cultures in a rotating wall vessel exhibit achange in sensitivity to drugs and toxins. Immortalized human renalcells were cultured for 24 hours in either a monolayer (diamonds), as 3Dcultures in a bag with beads for support (squares), or as 3D cultures ina rotating wall vessel (triangles), and treated with 0.05, 0.5, 5 or 50mg/ml throughout the time period of the culture.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present invention relates to the growth of cells in a suspensionculture that due to low shear and low operational turbulence minimizesmechanical cell damage and allows cells to associate intothree-dimensional structures, thereby promoting cellulardifferentiation. In an aspect of the invention, the three-dimensionalcell structures (or 3-D aggregates) are used to study microbialinfectivity from the perspective of the host-pathogen interaction. Inanother aspect of the invention, the three dimensional cell structuresare used to study the chemosensitivity of the 3-D tissue mass tochemotherapeutic agents and toxins.

The present invention further relates to the use of a bioreactor for thegrowth of cells in a suspension culture, wherein the shear levels aredefined. Specifically, cell culture is carried out in the bioreactorunder conditions of applied shear, Coriolis forces applied, with cellsfalling at a terminal velocity. This tissue culture approach permits thegeneration of 3-D differentiated tissue-like assemblies which model manyaspects of in vivo human tissues, and thus offer a new approach forstudying microbial infectivity from the perspective of host-pathogeninteraction, and chemosensitivity of tissues to therapeutic agents andtoxins.

Recent work with cultured epithelial cell lines has begun to elucidatethe molecular mechanisms by which microbial pathogens like Salmonella,induce inflammation. While the exact model by which Salmonella triggersan infection remains incompletely-defined, results from both in vivo andin vitro studies suggest that, in response to Salmonella invasion,epithelial cells rapidly upregulate the expression and secretion of anyarray of cytokines known to be important for the initiation of an acuteinflammatory response, including TNF-α and an array of proinflammatorymediators, like interleukin-8 (IL-8) and pathogen-elicited epithelialchemoattractant (PEEC), that chemoattract neutrophils and mononuclearphagocytes to the site of infection. Thus, in the early stages followingSalmonella invasion, intestinal epithelial cells (for e.g., Int-407)produce mediators that have the potential to orchestrate the onset ofthe mucosa inflammatory response.

It is possible that the increased expression of the proinflammatorycytokines TNF-α, and IL-6 by the Int-407 monolayers as compared to the3-D aggregates in response to Salmonella infection is, in part,responsible for the dramatic increase in damage to the monolayers ascompared to 3-D Int-407 tissue aggregates. Furthermore, the enhancedbasal level of expression of TGF-β1 by the uninfected 3-D Int-407 cellsas compared to monolayers is relevant to the in vivo condition, in whichthe intestinal mucosa constitutively produces high levels of thiscytokine.

Int-407 monolayers respond to infection with Salmonella by increasingthe transcriptional expression of genes encoding the proinflammatory andimmunomodulatory cytokines IL-1α, IL-1β, IL-6, and TNF-α, the latter ofwhich has been shown to induce apoptosis in several cell types. Incontrast, 3-D Int-407 tissue aggregates infected with Salmonellaincreased expression of the antiinflammatory cytokine IL-1Ra. Theseresults suggest that the relatively undifferentiated Int-407 monolayersreact differently to infection with Salmonella than do the 3-D Int-407tissue aggregates. Although infection of 3-D Int-407 tissue aggregateswith Salmonella stimulated increased transcription of these samecytokines, the magnitude of induction of expression was markedly less ascompared to Int-407 monolayers.

In vitro and in vivo studies have reported that, following infectionwith Salmonella, increased prostaglandin synthesis by PMNs andepithelial cells is important for the increased fluid secretion fromintestinal epithelium. However, the role of prostaglandin in thedevelopment of Salmonella-induced diarrheal disease in humans has notbeen established. It has been suggested that, in addition to regulatinggastrointestinal fluid secretion, epithelial-derived prostaglandin maylimit the extent of mucosa injury following infection with invasivebacteria. While it is unclear as to how prostaglandin may exertmucosa-protective events, they have been shown to down-regulate theproduction of several proinflammatory cytokines, such as IL-1. Theresults from the studies are in agreement with a role for prostaglandinin mediating a protective response against Salmonella-induced damage tothe epithelial mucosa. The constitutive levels of is PGE₂ synthesis inuninfected 3-D Int-407 cells is significantly higher than that observedfor uninfected monolayer cultures.

In an aspect of the present invention, methods for utilizing a form ofoptimized suspension culture to examine the infectivity of pathogenicorganisms and agents in cells and tissues are provided. Also providedare methods using a RWV to predict the chemosensitivity of cells andtissues to toxins and chemotherapeutic agents.

The invention is now described in detail with reference to specificembodiments. It will be understood that variations, which arefunctionally equivalent, are within the scope of this invention. Variousmodifications of the invention in addition to those shown and describedherein will be apparent to those skilled in the art from the descriptionherein and the accompanying drawings. Such modifications are intended tofall within the scope of the claimed invention.

The present invention encompasses methods for modeling infectiousdisease and chemosensitivity in cells and tissues. In an embodiment ofthe present invention, three-dimensional tissue masses are cultured inbioreactors or microgravity vessels, which provide low shear andessentially no relative motion of the culture environment with respectto the walls of the culture vessel.

In a further embodiment of the invention, tissues are grown in arotating wall vessel (RWV) under conditions designed to allow theformation of three-dimensional cell aggregates rather than monolayers,and promote the cellular expression of special features and propertiesnormally displayed in vivo. These three-dimensional tissue aggregatesserve as a model for microbial infectivity by a bacterial pathogen andas a model to test chemosensitivity.

One aspect of the invention involves the co-culturing in a rotating wallvessel of tissue constructs and microorganisms to facilitate analysis ofmicroorganism adhesion, invasion, virulence and growth. Themicroorganisms are placed in a RWV with culture medium along with tissueconstructs, and cultured for a period of hours to days, after whichparameters of the microorganism's effect on the tissues are assayed.These effects include microorganism growth, adherence, invasion, biofilmformation, cell toxicity and/or death. It has been proposed that up to60–70% of all infectious diseases are mediated at some point in theirtime course by the formation of a microbial biofilm. The inventionmodels the infectivity of tissues in vivo more accurately than monolayercultures. Dramatic differences occur between cells cultured in a RWVcompared to monolayer cultures in response to infection with amicroorganism, including differences in adherence, invasion, apoptosis,cytokine expression, prostaglandin synthesis and tissue pathology.Hence, cultivation of host tissues and cells with natural pathogens inthe RWV allows maintenance of both the differentiated features of thehost tissues and the natural infectivity of the microorganisms.

In a separate aspect of the invention, the three dimensional aggregatescan be infected with microorganisms outside the bioreactor environment.Specifically, the three dimensional aggregates are formed within thebioreactor, followed by seeding and propagation of the 3-D tissueconstructs in standard tissue culture systems, after which the 3Dtissues are infected with the pathogen.

An embodiment of the present invention provides a method of studying theinfectivity of a pathogen in tissues comprising the steps of:

isolating host cells;

placing said isolated host cells into a rotating wall vessel comprisingculture media;

applying sedimental shear stress to the cells in the cell culture toform a three-dimensional tissue mass;

introducing an infectious pathogen into said culture containing saidthree-dimensional tissue mass; and

assaying the infectivity of said infectious pathogen.

A further embodiment of the present invention provides a method ofstudying the infectivity of a pathogen in tissues comprising the stepsof:

isolating intestinal epithelial cells;

placing said intestinal epithelial cells into a rotating wall vesselcomprising culture medium;

applying sedimental shear stress to the cells in the cell culture toform a three-dimensional tissue mass;

seeding the formed tissue mass in a tissue culture vessel; and,

introducing an infectious pathogen to the formed tissue mass.

The above embodiments can optionally comprise the use of a culturematrix that would permit the adherence of the cells in the culture.Examples of such culture matrices consist of microbeads ormicrocarriers, including but not limited to, collagen-coated microbeadsor polymer scaffolding. Furthermore, the infection of the 3D tissue masswith the microorganism and the chemosensitivity assays may be carriedout either within the bioreactor or external to the bioreactor, understandard tissue culture conditions. Additionally, in a separateembodiment of the invention, an infecting microorganism is culturedeither in the bioreactor or under standard laboratory conditions, priorto the introduction of host cells.

Another embodiment of the present invention provides a method ofmeasuring the chemosensitivity of tissues to a toxic materialscomprising:

isolating host cells;

placing said isolated host cells into a rotating wall vessel comprisingculture medium;

applying sedimental shear stress to the cells in the cell culture toform a three-dimensional tissue mass;

introducing a toxic material into said culture containing saidthree-dimensional tissue mass; and

assaying the chemosensitivity of said toxic material.

In an embodiment of the present invention, the toxic material is achemotherapeutic material. In a further embodiment of the invention, thechemotherapeutic material is an antibiotic, more preferably gentamicin.

The present invention has a wide application for the modeling ofinfectious disease. Host tissues to be analyzed in the RWV infectivityexperiments include, but are not limited to, epithelial tissuesincluding those lining the gastrointestinal tract, as well as tissuefrom lung, kidney and liver. The infectivity of a number of pathogenicorganisms can be analyzed including viruses, bacteria, protozoans,parasites and fungi. More specifically, the pathogens used includestrains of Salmonella typhimurium, Escherichia coli, Shigella, Yersinia,campylobacter, Vibrio and other microbial pathogens like Listeriamonocytogenes. Application of the invention includes the identificationof candidate virulence genes, virulent serotypes for vaccine productionand other therapeutic strategies, analysis of host responses tomicrobial infection, as well as in the in vitro demonstration of theefficacy of vaccines.

Another aspect of the invention involves the culturing of tissues andcells in a RWV as a method to measure the chemosensitivity of theculture to toxins and chemotherapeutic substances. Cells are cultured inthe RWV for a period of time and then a test compound is introduced tothe growing cells. There is a dramatic change in the sensitivity ofcells and tissues cultured in a RWV to drugs and toxins. The inventiondemonstrates that cell aggregates or tissue explants grown in the RWVhave properties characterized by a change in the dose of drugs ortoxins, which cause cell changes such as apoptosis or necrosis. Thepresent invention permits the realistic modeling of the in vivosensitivity of a wide variety of cells including, but not limited tolung, kidney, liver, intestine and bladder. Application of the inventionincludes the development of protective agents for tissue toxicity,prediction of the response of patient specific cancers tochemotherapeutic agents and prediction of the toxic effects oftherapeutics.

In humans, S. enterica serovar Typhimurium is among the most commonSalmonella serotypes isolated from humans suffering from infectiousgastroenteritis, and has long been recognized as a major public healthproblem. Gastroenteritis results from infection of the small intestineafter ingestion with Salmonella. Following ingestion, Salmonella adheresto and invades into both the M cells of the Peyer's patches and theintestinal enterocytes. This ability to attach to and penetrateintestinal epithelium is an essential feature in the pathogenicity ofSalmonella infection. The initial interactions between Salmonella andthe host intestinal epithelium are believed to play a key role inmediating the intense inflammatory and secretory response which is ahallmark of serovar Typhimurium infections in humans. Both in vitro andin vivo studies have shown that the inflammatory response to Salmonellainfection is characterized by an influx of polymorphonuclear leukocytes(PMNs) into the intestine and lumen resulting in a net increase in fluidsecretion into the lumen. Studies with cultured intestinal epithelialcells have shown that, shortly after contact, Salmonella engages hostcells in a complex biochemical cross-talk, which triggers host-cellsignal transduction pathways ultimately resulting in host cytoskeletonrearrangement, cell membrane ruffling, bacterial uptake, and productionof prostaglandins, and proinflammatory cytokines.

RWV bioreactors have been used to develop three-dimensional cultures ofhuman intestinal epithelial cells which more accurately model thebehavior of in vivo tissues as compared to monolayer cultures of thesame cells, and thus would be predicted to more closely replicate thecomplex environment encountered by Salmonella during the natural courseof human infection. The present invention that the 3-D intestinal cellsreact much differently to Salmonella infection as compared toconventional monolayers of the same cell line, including differences inbacterial adherence and invasion, apoptosis, cytokine expression,prostaglandin synthesis, and tissue pathology.

The following is a description of culturing and growing a representativebacterial strain, which can be used to practice the present invention.Representative infectivity studies were performed using wild-typeSalmonella enterica serovar Typhimurium, χ3339, which is an animalpassaged isolate of the virulent SLI344 wild-type. See Gulig andCurtiss, Infection and Immunity, 55: 2891–2901 (1987), which is fullyincorporated by reference herein. Bacterial cells were first grown inLennox broth (L-broth) as static overnight cultures at 37° C. Cultureswere then inoculated at a dilution of 1:200 into 50 ml of L-broth andsubsequently grown with aeration at 37° C. until reaching mid-log phaseof growth. Unless otherwise stated, all infectivity studies wereperformed at a multiplicity of infection (MOI) of between one-to-10bacteria per host cell.

The following is a description of the development of a three-dimensionaltissue model. The human embryonic intestinal epithelial cell lineInt-407 was obtained from the American Type Culture Collection (ATCC)(CCL-6) and was initially grown in Corning T-75 flasks (2×10⁵ cells/ml)at 37° C. in a 5% CO₂ environment in preparation for seeding into theRWV. The cells were cultured in a specialized growth medium comprised ofa triple-sugar minimal essential medium-alpha/L-15 base supplementedwith 6% fetal bovine serum (FBS), designated GTSF-2. After 24 hours, thespent media was removed, fresh GTSF-2 was added, and the cells werecultured until reaching approximately 70% confluency. Cells were thenwashed once with pre-warmed calcium-and magnesium-freephosphate-buffered saline (PBS), removed from the flask by treatmentwith 0.25% trypsin, and resuspended in fresh medium at a density of2×10⁵ cells/ml. The cells were assayed for viability by trypan blue dyeexclusion. Cells were then introduced into the RWV (Synthecon, Inc.)containing 5 mg/ml Cytodex-3 microcarrier beads (Pharmacia), resultingin a final ratio of 10 cells/bead. Cytodex-3 microcarriers were Type 1,collagen-coated dextran beads (average diameter size 175 μm). Cells werecultured in the RWV bioreactors in GTSF-2 at 37° C. and 5% CO₂ withinitial rotation at 20 rpm. Rotation speed was increased throughoutgrowth to maintain cell aggregates in suspension. Cell growth wasmonitored daily by measurements of pH, dissolved CO₂ and O₂ and glucoseutilization using a Corning Blood Gas Analyzer (Model 168) and a BeckmanGlucose Analyzer 2, respectively. Fresh medium was replenished by 90% ofthe total vessel volume each 12–24 hr, depending upon the growth rate ofthe cultures. As metabolic requirements increased, fresh medium wassupplemented with an additional 100 mg/dl of glucose. Immediately priorto use of the 3-D intestinal tissues, fresh medium was added to thecultures. For all studies, 3-D intestinal tissues were cultured in theRWVs for 28–32 days prior to their use.

Int-407 cells cultured as 3-D aggregates or as monolayers were subjectedto immunohistochemical characterization. Samples forimmunohistochemistry were taken from multiple experiments, fixed, andsectioned using method well known to one of ordinary skill in the art.Immunophenotyping was performed on 0.5 μm-thick sections of the 3-DInt-407 tissue aggregates, or monolayer culture controls, afterextraction of the epoxy with melting solution for light microscopystudies. Slides of sectioned material were subsequently rehydrated andsubjected to immunoperoxidase staining with panel of antibodies. Toevaluate the mucin content, specimens were stained with periodicacid-Schiff (PAS).

Morphological comparisons between normal human small intestine, Int-407monolayers, and 3-D Int-407 aggregates were determined from paraffinembedded sections of these samples stained with hematoxylin-eosin andanalyzed via light microscopy.

For scanning electron microscopy (SEM) analysis, 3-D cell and monolayersamples were fixed in 4% glutaraldehyde. The samples were then immersedin 0.1% osmium tetroxide solution (Electron Microscopy Sciences) anddehydrated in a graded alcohol series to 100% ethanol. The 3-D cellaggregates were placed on a glass cover slip treated with 1%polyethyleneimine. Both 3-D cell cultures and monolayers were chemicallytreated with hexamethyldisilazane (Electron Microscopy Sciences) andallowed to dry. All cells were then mounted on specimen stubs andsputter coated with gold-palladium for observation in a JEOL 660T orPhillips XL-30 series scanning electron microscope.

For confocal imaging, samples were prepared as described using methodswell know to one of ordinary skill in the art. Confocal imaging wasperformed using a Zeiss LSM 400 series laser scanning microscope.Fluorochrome excitation of Alexa 488 labeled secondary antibodies was by488 nm filtered emission from a Kr-Argon laser source. Each image is thecompilation of 16 scans of 2 seconds each, collected at identicalexposure levels (the confocal pinhole size, laser output, filtersettings and gain and contrast settings were held constant throughout).Each scan was corrected for background variations and noise by the LSMscanning software during the compilation process. This correctionresulted in enhanced resolution, but did not affect exposure levels.Post-collection processing of the images was performed using AdobePhotoShop. As before, no changes in image brightness or contrast weremade.

For transmission electron microscopy (TEM) analysis, samples wereprepared by methods well known to those of ordinary skill in the art.TEM imaging was performed using a JOEL 1010 series transmission electronmicroscope.

Adherence and invasion assays were conducted using methods well known toone of ordinary skill in the art, with the modification that 3-D Int-407cells were seeded into 24-well tissue culture plates for infectivityassays. The number of cells associated with 3-D aggregates weredetermined. In an aspect of the invention, the infectivity assays arecarried in the bioreactor or RWV, by co-culturing the tissue constructsand microorganisms.

The cytokine mRNA profiles expressed following Salmonella infection of3-D Int-407 tissue aggregates and Int-407 monolayers were analyzed andquantified using a commercially available multiprobe RNAse protectionassay (RiboQuant, PharMingen, San Diego, Calif.). Total RNA isolatedfrom uninfected and infected 3-D intestinal aggregates and Int-407monolayers was extracted using TriReagent in an acid guanidiniumthiocyanate-phenol-chloroform method according to the manufacturer'sinstructions (Molecular Research Center, Inc., Cincinnati, Ohio). TotalRNA was then used in RNAse protection assays with a mixture of[³²P]UTP-labeled antisense riboprobes generated from a panel ofdifferent human cytokine templates, specifically in this case, TNF-α,IL-1α, IL-1β, TNF-β, lymphotoxin-β IFNγ, IFNα, IL-6, IL-10, IL-12, IL-18and TGF-β1. The template mixtures containing these cytokine templatesalso included templates for the housekeeping genes encodingglyceraldehyde-3-phosphate dehydrogenase (GAPDH) and L32 (a ribosomalprotein) to ensure equal loading of total RNA onto 6% polyacrylamide/7Murea gels.

Quantification of each mRNA was accomplished with a Fuji FLA-3000Phosphorimager and ImageGauge software. Each mRNA band was normalized toL32 or GADPH from the same RNA sample and the data presented asarbitrary units of each given cytokine.

In a prostaglandin immunoassay, levels of PGE₂ were determined inculture supernatants of infected and uninfected 3-D Int-407 cells seededinto 24-well tissue culture plates, and Int-407 monolayers by enzymeimmunoassay according to the manufacturer's instructions (R&D Systems).Bacteria were prepared for infections as described above with an MOI ofbetween 50:1 to 100:1. All Int-407 cells were infected for 1 h withχ3339, and cultured for an additional 7 h in the presence of gentamicin(10 μg/ml) to kill any remaining extracellular bacteria. Culturesupernatants were sampled for PGE₂ expression prior to addition ofSalmonella, and at 1, 2 and 8 h following infection.

Following incubation for 1.5 hours with or without Salmonella, Int-407monolayers and 3-D aggregates were dissociated into individual cells bytreatment with 0.1% EDTA for analysis of apoptosis. The cell number andviability were determined by trypan blue dye exclusion. The resultingsingle cell suspensions were washed with calcium-containing PBS andincubated for 15 minutes with FITC-conjugated Annexin V (R&D Systems)and/or propidium iodide to quantitate apoptosis and necrosis,respectively. Positive controls for apoptosis and necrosis were culturesof 3-D Int-407 tissue aggregates or monolayers cultured with actinomycinD (1 μg/ml) and sodium azide (1.0%), respectively. These control cellswere incubated with actinomycin D or sodium azide for 2–3 h prior towashing and staining with FITC-Annexin V and propidium iodide andanalysis on a FACSVantage flow cytometer.

Investigations into the interaction of Salmonella with the humanintestinal epithelium have been limited by the lack of in vitro and invivo models which faithfully replicate the in vivo condition. Inparticular, conventional tissue culture technology has failed to yieldhigh-fidelity, multicellular three-dimensional models of intestinalepithelium which are suitable for investigations into Salmonella-inducedgastroenteritis. A class of rotating bioreactors was used, which areoptimized to minimize shear and turbulence, to culture 3-D humanintestinal epithelial tissue retaining many differentiated features as amodel to study the infectivity of the enteroinvasive pathogenSalmonella. The 3-D cultured human intestinal cells more accuratelymodel in vivo tissues as compared to monolayer cultures of the samecells, and thus more closely replicate the complex environmentencountered by Salmonella during the natural course of human infection.These observations are characterized by dramatic differences between the3-D Int-407 cells and Int-407 monolayers in response to infection withSalmonella, including differences in adherence, invasion, apoptosis,prostaglandin synthesis and tissue pathology.

Immunohistochemical characterization of numerous histological sectionsof 3-D Int-407 tissue aggregates and Int-407 monolayers by fluorescentmicroscopy were consistent with the degree of differentiation betweenthe two cultures. In general histological sections of the 3-D intestinalaggregates revealed tissue organization and differentiation similar tothat found in vivo, while monolayer material showed poor organization,reduced expression of specific markers normally present indifferentiated human intestinal epithelium, and reduced expression ofthe differentiated phenotype. The increased express by 3-D aggregates ofextracellular and basement membrane proteins, specific epithelial andendothelial cell markers, and mucin, indicates important aspects ofdifferentiated tissues, and suggests that the 3-D cell aggregates havethe ability to organize and develop into complex tissue assembliescapable of expressing in vivo-like functional characteristics. Theenhanced expression by 3-D aggregates of sialyl Lewis A suggests thatthe M-cell glycosylation pattern expressed on the surface of the 3-DInt-407 aggregates grown in the RWV may present an epithelial surface tothe infecting microbe which more closely resembles that encounteredwithin the host during infection, relative to the Int-407 monolayers.This finding is of particular relevance, since numerous pathogens inaddition to Salmonella demonstrate a tropism for M cells. However, nocontinuous M cell line is currently available.

The expression of distinct glycoconjugates by normal human Peyer's patchM cells suggests an important role for carbohydrate epitopes in thefunction of this unique epithelial cell type. The structuralmodifications of the M-cell apical surface and the display of particularoligosaccharides together would allow M cells to present a conspicuouslyunique biochemical face to the lumen which might facilitate adherence,uptake, and immunological sampling of microorganisms. Expression ofcytokeratin 18, a marker associated with tumor, abnormal andundifferentiated cells, was dramatically down-regulated in the 3-Daggregates as compared to monolayers. The decreased expression ofcytokeratin 18, is suggestive of a shift from a relativelyundifferentiated state (as in the monolayers) to the differentiatedphenotype observed for the 3-D tissue aggregates. Histochemical stainingwith a periodate-Schiff (PAS) assay demonstrated that the 3-D constructscontained a mucous-producing sub-population of cells that expressed adifferential phenotype compared to the surrounding cells. Thisdifferential staining pattern is very similar to that observed formucous-secreting goblet cells within fixed, paraffin sections ofintestinal tissue. In contrast to the 3-D aggregates, the monolayers didnot produce a positive PAS reaction. The presence of a sub-population ofPAS-positive cells in paraffin sections of the intestinal-derivedInt-407 cell constructs is highly suggestive that culture of these cellsin the bioreactor as three-dimensional, multi-cellular constructsresults in the expression of differential cellular phenotypes, one ofwhich apparently is a “mucous-producing” cell type and representsanother important similarity to the intestinal mucosa.

Studies of the intestinal 3-D aggregates and monolayer cultures viawhole mount confocal microscopy using proteins that serve as widelyaccepted markers of epithelial differentiation revealed a number ofimportant distinctions between the two cultures conditions. While bothculture types expressed epithelial specific antigen (ESA) and cadherinon the cell surfaces, the stratified epithelioid 3-D constructs expressa much greater amount of these proteins. Furthermore, the proteins arelocalized specifically to cell-cell interfaces within the closelyadherent cell layers of 3-D cultures. This finding indicates that the3-D culture constructs have well-defined lateral cell-cell borders andjunctional complexes throughout, while the monolayer cultures appear topossess nascent lateral polarity in a large proportion of the cellpopulation.

In comparison to Int-407 cells grown as monolayers in culture, theexpression of the basal lamina proteins Type IV collagen and lamininwere dramatically upregulated in the 3-D cultures. During thedevelopment of polarity in the epithelial phenotype, the basal lamina isdeposited at the cell-substrate interface of a nascent epitheliumfollowing the establishment of strong lateral polarity within theepithelium. In 3-D Int-407 cultures, which already possesswell-established lateral polarity, the upregulated synthesis of basallaminal proteins is highly indicative of the first stages in theformation of apical-basal polarity. Furthermore, the deposition of theseproteins at the cell-substrate interface, in this case the surface ofthe cytodex microcarrier beads, serves to convince us that the 3-Dcultured cells are setting up apical-basal polarity by constructing anascent basal lamina on their substrate.

Moreover, unlike their monolayer counterparts, analysis of Int-407samples by TEM indicated that the 3-D intestinal aggregates exhibitedwell-formed and numerous microvilli at their apical cell surfaces,abundant and well-developed vacuolar-like formation, and extensive andwell-formed tight junctions. In summary, the results of microscopystudies demonstrate that, based upon biochemical differentiation, cellpolarity and cell ultrastructure, the 3-D Int-407 aggregates much moreclosely resemble human differentiated intestinal epithelium than doconventional monolayer cultures of the same cells. As such, 3-D Int-407cultures would be predicted to more closely replicate the complexenvironment encountered by Salmonella or other pathogen during thenatural course of human infection.

Results from infectivity studies demonstrated that Salmonellaestablished infection of the 3-D human intestinal cells in a muchdifferent manner as compared to monolayer cultures. Salmonella exhibitedsignificantly reduced abilities to adhere to and invade the 3-D humanintestinal aggregates as compared to monolayer cultures. In agreementwith this finding SEM analysis of Salmonella-infected intestinal cellsrevealed fewer bacteria associated with the surface of the 3-Daggregates as compared to monolayers following time-course infections.This may be a reflection of the differential expression of host cellsurface adhesions by the 3-D Int-407 cells which are more relevant tothe in vivo setting as compared to growth as monolayers. AlthoughSalmonella adhered to and invaded the 3-D intestinal cells poorly, thesecells did exhibit signs of Salmonella induced-damage at laterpost-infection time points. The question arises as to how Salmonella isable to induce damage to the host epithelium if it adheres and invadespoorly to this tissue. In vitro studies have shown that the Type IIIsecretion system encoded on Salmonella Pathogenicity Island I isrequired for the translocation of proteins into host epithelial cellsand the induction of fluid secretion, and inflammatory responses. Giventhe inherent differences in the structural modifications of the surfacesof the 3-D intestinal epithelial cells as compared to monolayers, it ispossible that the secretion of Type III effector proteins is induced ina different manner in the 3-D aggregates following Salmonella infection.

Both in vitro and in vivo studies have shown that interaction ofwild-type virulent serovar Typhimurium with intestinal epitheliuminduces a number of morphological changes to the host epitheliumimportant for subsequent bacterial invasion. These changes include,cytoskeletal rearrangement with the formation of membrane ruffles uponthe site of Salmonella contact, blunting and transientdenuding/degeneration of microvilli from enterocytes, destruction of Mcells, and the formation of pathological lesions. 3-D Int-407 aggregatesdisplayed minimal change in overall morphology following Salmonellainfection as compared to the extensive loss of integrity observed forInt-407 monolayers following the same time course of infection. Sincethe 3-D aggregates more closely resemble human intestinal epithelium,the difference in structural integrity following Salmonella infection ofthese tissues as compared to monolayers is more reflective of an in vivoinfection.

Induction of tissue damage in the form of apoptosis is a common responseof host tissues to infection with bacterial pathogens. Several mediatorsproduced in response to Salmonella infection, such as TNF-α, also havethe potential to induce apoptosis of epithelial cells. Not surprisingly,Salmonella species have been shown to induce apoptosis followinginfection of several cell types, including cultured macrophage and ahuman colonic epithelial cell line. However, the onset of cell apoptosisin each of these cell types following Salmonella infection wasdramatically different, with a rapid onset of apoptosis (within 2 hours)following infection of macrophage and a delay of up to 12–18 hoursfollowing bacterial entry into colon epithelial cell lines. Thepercentage of apoptosis in Int-407 cells grown as monolayers wassignificantly increased 90 minutes post-infection with Salmonella ascompared to uninfected controls. There was a rapid onset of apoptosisfollowing Salmonella infection of Int-407 monolayers, with between a70%–90% apoptotic index occurring 90 minutes after infection. However,there was no difference in apoptosis between infected and uninfected 3-DInt-407 tissue aggregates at the same time post-infection. In addition,the results from adherence and invasion studies also support theseobservations, as Salmonella adherence to and invasion into 3-D Int-407aggregates was significantly less than that observed for Int-407monolayers. Considering that the vast majority of cases ofSalmonella-induced gastroenteritis go unreported, (less than 5%), itseems unlikely that following ingestion of Salmonella, approximately 70%of human intestinal epithelial cells undergo apoptotic death. Thus, thelower levels of apoptosis observed following Salmonella infection of 3-Dhuman intestinal aggregates would likely be more reflective of an invivo infection.

The present invention discloses the use of three-dimensional tissueaggregates cultured in the RWV as a model for microbial infectivity by abacterial pathogen. The bacterium used in these studies, Salmonellaenterica serovar Typhimurium, belongs to the family Enterobacteriaceae,a larger group of Gram negative rods whose natural habitat is theintestinal tract of humans and animals, and which includes most of thebacteria that cause intestinal and diarrheal disease, considered to beone of the greatest health problems globally. However, it should benoted that there are many bacterial pathogens which gain access toanimal hosts in a manner similar to Salmonella, i.e., by attaching toand invading the gut-associated lymphoid tissue (GALT) and thendisseminating to visceral lymphoid tissues to cause systemic disease.These bacteria include many other enteric pathogens, such as Shigellaand enteropathogenic E. coli, including the 0157:H7 type, as well asother microbial pathogens such as Listeria monocytogenes. Thus, the useof 3-D tissue aggregates of human intestinal cells will have broadapplicability and therefore significance in detailing how microbialpathogens which are invasive through the GALT gain access to andestablish infection within the host.

Characterization of the in vivo epithelial cell expressioncharacteristics of the RWV-grown intestinal 3-D aggregates relative tomonolayer cultures was determined by examining immunohistochemical andproto-oncogene expression patterns of histological sections of thesecultured cells. Immunohistochemical analysis was performed onhistological sections of tissue obtained from the RWV and monolayercontrols with antibodies against collagen II and III, fibronectin,vimentin, pancytokeratin, von Willebrand factor, sialyl Lewis A, villin,and cytokeratin 18. Immunohistochemical analysis of 3-D Int-407aggregates as compared to monolayers revealed striking differencesbetween the two cultures. In general, the 3-D intestinal aggregatesdemonstrated tissue organization similar to that found in vivo (FIGS. 1Cand 1A, respectively), while monolayer material showed poor organization(FIG. 1B), reduced expression of specific markers normally present indifferentiated human intestinal epithelium, and reduced expression ofthe differentiated phenotype. Specifically, 3-D aggregates of Int-407cells exhibited very strong staining for the extracellular matrixproteins collagen type II (FIG. 2B) and fibronectin (FIG. 2D), ascompared to weakly to moderately positive staining for these sameepitopes in Int-407 Monolayers (FIGS. 2A and C, respectively). Inaddition, expression of the M-cell glycoconjugate sialyl Lewis A antigenwas also enhanced in the 3-D aggregates (FIG. 2F) relative to themonolayers (FIG. 2E). Three-dimensional aggregates also exhibitedincreased staining for the cytoskeletal marker villin (an abundantprotein in the brush border epithelial cells of the small intestine)(FIG. 2H), as compared to the weaker staining for this epitope observedin Int-407 monolayers (FIG. 2G). Expression of cytokeratin 18, a markerassociated with tumor, abnormal and undifferentiated cells, wasdramatically down-regulated in the 3-D aggregates (FIG. 2J) as comparedto monolayers (FIG. 2L). Moreover, the 3-D aggregates also exhibitedenhanced staining for von Willebrand factor (endothelial cell marker),collagen III, vimentin, and pancytokeratin as compared to monolayers(data now shown). Negative controls for all immunohistochemical analysesare shown in FIGS. 2K (monolayers) and 2L (3-D aggregates),respectively. In addition, histochemical staining with aperiodate-Schiff (PAS) assay showed mucins to be produced by the 3-DInt-407 aggregates (FIG. 3), but not by monolayers (data not shown).

Characterization of the in vivo epithelial cell expressioncharacteristics of the intestinal 3-D aggregates relative to monolayercultures was further studied via immunohistochemical analysis by wholemount confocal microscopy of tissue obtained from the bioreactor andmonolayer controls with antibodies against type IV collagen, laminin,cadherin, epithelial specific antigen (ESA) and cytokeratin 7, all ofwhich are proteins that serve as widely accepted markers of epithelialdifferentiation. Immunostaining for epithelial specific antigen (ESA),an antigen expressed only in epithelia, indicated that the Int-407 cellswere epithelial in nature. All cells in both the monolayer cultures andin the 3-D epithelial constructs expressed this antigen (FIGS. 4B and A,respectively). Confocal microscopy of the cell surface ESA showed howdensely populated the interstices of the stratified epithelioid 3-Dconstructs were with closely adherent layers of cells, in contrast tothat seen in monolayer culture (FIGS. 4A and B, respectively).

As epithelia form, the cells adhere to each other via lateral cell-cellinteractions mediated by a class of plasmalemmal binding proteins knownas the cadherins. Developmentally mature epithelia display a very tightassociation of cadherins to their lateral surfaces in the areas wherethey adhere to other cells. Ultrastructurally, this zonulae adherens iseasily identified as a band of actin filaments decorated with denseaccumulations of cadherin protein. The 3-D Int-407 cultures showed justsuch a dense, regular appearance of the cadherin assemblies along theirlateral cell-cell boundaries (FIG. 4C). By comparison, the monolayercultures displayed copious cadherin accumulations only in areascontaining closely proximated cells, areas that occur with much lessfrequency (FIG. 4D). This indicated that the 3-D culture constructs havevery well-defined lateral cell-cell borders and junctional complexesthroughout, while the monolayer cultures appear to still be in theprocess of initiating lateral polarity in a large proportion of the cellpopulation.

In comparison to Int-407 cells grown as monolayers in culture, theexpression of laminin (FIGS. 4I–J) and Type IV collagen (FIGS. 4K–L)were dramatically upregulated. Both of these proteins are known tocontribute to formation of the basal lamina, a structural component ofall developmentally mature and fully functional epithelia.Developmentally, the basal lamina is deposited at the cell-substrateinterface of a nascent epithelium defined only by lateral cell-celladhesions, thus imparting an apical-basal polarity to the entirestructure. In 3-D Int-407 cultures, the presence of these basal laminalproteins, and their deposition at the cell-substratum interface, ishighly indicative of the first stages in the formation of apical-basalpolarity (FIGS. 4I and K, respectively).

The expression and histological pattern of a group of cytoskeletalproteins, the cytokeratins, was studies. Cytokeratin 19, a markerassociated with tumor and abnormal cells, was dramaticallydown-regulated in the 3-D aggregates (FIG. 4E) (note: 150 μm beads aredelineated by a dotted outline) as compared to monolayers (FIG. 4F).Conversely, cytokeratin 7, absente in the monolayer cultures (FIG. 4H)was upregulated by 3-D culture conditions (FIG. 4G). The modulation ofcytokeratin is indicative of large-scale phenotypical alterationsassociated with 3-D culture.

Characterization of the in vivo epithelial cell expressioncharacteristics of the 3-D intestinal aggregates relative to monolayercultures was further examined using TEM analysis. Overall, monolayercultures demonstrated poor structural and functional fidelity ascompared to their 3-D counterparts. Specifically, monolayer culturesexhibited irregular microvilli development (FIG. 5A), significantcellular granularity (FIG. 5B) accompanied by minimal tight junctionformation (FIGS. 5A, B), poorly formed gladular/vacuolar-like formation(FIG. 5B), and lack of apical polarity. In contrast, 3-D culturesdemonstrated well-developed and abundant microvilli (FIG. 5C) which wereapically oriented (FIG. 5F), well-formed tight junctions (FIGS. 5D, E),and numerous, well-formed vacuolar-like structures (FIG. 5D).

An initial step in the pathogenesis of Salmonella infection is theadherence and entry of these organisms into the intestinal epithelium.Accordingly, the ability of serovar Typhimurium χ3339 to adhere to andinvade 3-D tissue aggregates of Int-407 cells was examined, as comparedto Int-407 monolayers. Representative data from three trials showedthat, with respect to the percentage of initial inoculum (MOI 10:1), theadherence and invasion of Salmonella into 3-D Int-407 cells (2.6±1.3 and1.3±0.8, respectively) was significantly lower than that observed forInt-407 monolayers (48.0±19.0, and 51.0±18.0, respectively).

Pathology of 3-D tissue aggregates relative to monolayers before andafter infection with Salmonella. Scanning electron microscopy (SEM) wasused to examine surface interactions and membrane structural alterationswhich occurred over time following Salmonella infection of 3-D Int-407aggregates as compared to monolayers. Observations of numerous samplesrevealed major changes in the integrity of the monolayers as compared to3-D intestinal tissues following time-course infections with Salmonellaat the same MOI. At time zero, SEM revealed typical, young confluentmonolayers (FIG. 7A). As early as 10 minutes post-infection, observabledifferences in structural integrity were observed in the Int-407monolayers as compared to uninfected controls, the former showingextensive formation of membrane blebs, and several areas of protrudingcytoplasm resembling membrane ruffles (FIG. 7B). One hour postinfection,the Int-407 monolayers appeared swollen with membrane blebs and theformation of pathological lesions (FIG. 7C). There was a dramatic lossof structural integrity of the monolayers at 2 h post-infection,including the presence of extensive lesions, as well as large swollenareas of denuded surface membrane (FIG. 7D).

Three-dimensional Int-407 tissue aggregates infected with Salmonella didnot display as extensive loss of structural integrity as observed forinfected Int-407 monolayers following the same time-course of infection.At time zero (i.e. uninfected control), SEM revealed dense masses ofcells associated with the microcarrier beads with apparent mucous-likeextracellular secretions covering the cells. Note the “furry” appearanceof what appear to be microvilli present on the epithelial cellaggregates (FIG. 8A). Ten min post-infection, the surface of the 3-DInt-407 cells appears slightly irritated, however, unlike the infectedmonolayers, few bacteria are observed in association with the surface ofthe 3-D aggregates (FIG. 8B). One hour post-infection, numerousdepressed/pitted areas are evident, which are devoid of mucous-likeextracellular secretions (FIG. 8C). By two hours post-infection,numerous lesions are apparent on the surface of the 3-D Int-407 cells.In addition, regions of membrane blebbing are also abundant (FIG. 8D).

In order to characterize the cytokine expression of 3-D Int-407aggregates as compared to monolayer cultures following infection withserovar Typhimurium, a commercially available multiprobe RNAseprotection assay was used to quantify and compare the cytokine mRNAprofiles. Infection of both Int-407 monolayers and 3-D aggregates withserovar Typhirnurium resulted in significantly increased expression ofTNFα, IL-6, IL-1α, IL-1β and IL-1RA at 1 hour and 2 hours afterinfection compared to uninfected monolayers and 3-D aggregates,respectively. Representative data are shown for TNFα (FIG. 9A) and IL-6(FIG. 9B). Infection of INT-407 monolayers with Salmonella inducedsignificantly increased expression of TNFα at one hour after infection(P<0.0005) and at 2 hours after infection (P<0.0001) compared touninfected monolayers (FIG. 9A). TNFα mRNA levels were significantlyelevated at one hour (P<0.0005) and 2 hours (P<0.0001) after infectionof 3-D aggregates compared to uninfected 3-D aggregates, however, TNFα.expression did not increase from one hour after infection to two hoursafter infection (FIG. 9A). Although, infection of both Int-407monolayers and 3-D aggregates resulted in increased TNFα mRNA levels.TNFα expression was more than five-fold higher in the monolayers at twohours after infection compared to the 3-D aggregates. Similarly,infection with serovar Typhimurium induced expression of IL-6 in bothInt-407 monolayers and 3-D aggregates (FIG. 9B). Significantly higherlevels of IL-6 mRNA were detected at one hour (P<0.002) and two hours(P<0.0001) after infection of Int-407 monolayers compared to uninfectedmonolayers. Infection of Int-407 monolayers resulted in greater than a50-fold increase in IL-6 transcription at two hours after infectioncompared to uninfected monolayers (FIG. 9B). Although IL-6 transcriptionin Int-407 3-D aggregates was significantly higher at two hours afterinfection (P<0.005) compared to uninfected 3-D aggregates, the overallincrease was just over three-fold in magnitude.

Transcription of IL-1α. IL-1β and IL-1Ra was also elevated followinginfection of Int-407 monolayers and 3-D aggregates (data not shown). Ineach case, constitutive expression was higher in uninfected 3-Daggregates compared to monolayers. By two hours after infection ofmonolayers, IL-11 expression increased by 17-fold compared to uninfectedmonolayers and IL-1β expression increased by 13-fold; whereas, expressof the IL-1 inhibitor, IL-1Ra, increased by just over three-fold. Incontrast, two hours after infection of 3-D aggregates, IL-1α expressionincreased four-fold and IL-1β expression increased approximatelythree-fold while expression of IL-1Ra was increased two-fold compared touninfected 3-D aggregates. Taken together, it appears that IL-1α andIL-1β expression is upregulated more in Int-407 monolayers compared to3-D aggregates; whereas, expression of the IL-1 inhibitor, IL-1Ra, isincreased about the same in monolayers compared to 3-D aggregatesfollowing infection with serovar Typhimurium. Thus, proinflammatoryeffects of IL-1α and IL-1β may be more prominent in the Int-407monolayers following infection with serovar Typhimurium as compared tothe 3-D aggregates.

TGF-β1 frequently serves in an immunosuppressive role, and therefore,the expression of this cytokine following infection of Int-407monolayers and 3-D aggregates was examined. Although, infection withserovar Typhimurium did not significantly affect expression of TGF-β1 atone or two hours after infection of either monolayers or 3-D aggregates,TFG-β1 mRNA levels were always two-fold higher in the 3-D aggregatescompared to the monolayers. Assay for apoptosis of human intestinalepithelial cells after Salmonella infection. When grown as monolayers,several cell lines have been shown to undergo apoptosis followingSalmonella infection. To assess the relationship between bacterialinfectivity and death of human intestinal Int-407 cells cultured in theRWV or as monolayers, flow cytofluorometry was used to characterizeapoptotic cell death of these cells before and after Salmonellainfection. Following 1.5 hours infection with Salmonella. Int-407monolayers contained approximately eightfold more apoptotic cells(68.3%) than control uninfected monolayers (8.8%). In contrast, therewas no increase in apoptosis following the same time course ofSalmonella infection of 3-D Int-407 aggregates, with a 5.2% and 7.3%apoptotic index of aggregates pre and post-infection, respectively.

To determine whether there was a differential induction of prostagladinsynthesis in response to Salmonella infection between the Int-407 cellscultured as 3-D aggregates or as monolayers, PGE₂ levels in these cellswas measured by immunoassay before and after infection with χ3339 Levelsof PGE₂ were not increased in 3-D Int-407 cells or in monolayersfollowing Salmonella infection at 1, 2 or 8 h (Table 1). In contrast,there was a dramatic increase in the level of constitutive PGE₂synthesis (approx. 79×) in the uninfected 3-D Int-407 cells as comparedto the monolayer cultures (Table 1).

TABLE 1 PGE₂ production by Int-407 cells before and after infection withSalmonella¹ PGE₂ produced (ng/ml) Culture Control 1 hour 2 hours 8 hoursInt-407 237.1 ± 16.2  349.3 ± 84.7  180.6 ± 18.6  162.9 ± 124.0Monolayers 3-D Int-407 18,724.3 ± 1.590    16.985 ± 1,240.0 14,142.9 ±2,280   16,772.0 ± 73.6    Cells ¹Time course of PGE₂ levels before andafter Salmonella infection of 3-D Int-407 cells and Int-407 monolayers.Monolayers of Int-407 cells in 24 well plates, or 3-D Int-407 cellscultured in 24 well plates, were infected for 1 hour with serovarTyphimurium χ3339 at an MOI of approximately 50:1. After 1 h, cultureswere further incubated with gentamicin (10 μg/ml) for another 7 hours.At the indicated times after infection, culture supernatant wascollected and analyzed for PGE₂ by enzyme immunoassay.

In order to compare the chemosensitivity of cells in a monolayerrelative to those in a 3D aggregate either under static conditions, orfalling at terminal velocity with applied shear, immortalized humanrenal cells were cultured in DMEM/F12 medium with 10% calf serum for 24hours in either a Monolayer, a conventional cell bag culture or arotating wall vessel. In separate aliquots of cells in separate vessels,gentamicin was added throughout the time period of culture atconcentrations of 0.05, 0.5, 5 or 50 mg/ml. The cells in all cultureswere then treated with gentamicin for six hours and the number ofapoptotic and necrotic cells were counted using a flow cytometry assay.FIG. 10 depicts one data set for this experiment in which the monolayeris depicted as diamonds, the 3-dimensional cultures grown on beads in abag are depicted as squares, and the 3-dimensional cultures in therotating wall vessel are depicted as triangles. As judged byKogomorov-Smirnoff summation statistics, renal cell growth on beadsunder static condition increases basal cell death rates. However, onlythe use of a rotating wall vessel induced dose-dependent cell death inresponse to gentamicin. These data demonstrate directly that there is adramatic change in the sensitivity of cells to drugs and toxins in 3Dcell cultures.

Although preferred embodiments of the present invention have beenillustrated in the accompanying drawings and described in the foregoingdetailed description, it will be understood that the invention is notlimited to the embodiments disclosed, but is capable of numerousrearrangements, modifications and substitutions without departing fromthe spirit of the invention as set forth and defined by the followingclaims.

1. A method of studying infectivity of a pathogen in tissues comprisingthe steps of: isolating cells from a host tissue sample; placing saidisolated host cells into a bioreactor comprising culture medium to forma cell culture; applying sedimental shear stress to the cells in thecell culture to form a three-dimensional tissue mass; seeding the formedtissue mass in a tissue culture vessel; introducing an infectiouspathogen into said three-dimensional mass; and assaying the infectivityof said infectious pathogen, wherein said assaying comprises the stepsof: obtaining a measurement of at least one of the following parametersafter introducing said infectious pathogen: adherence of said infectiouspathogen to cultured cells; cytokine expression in cultured cells; orprostaglandin synthesis in cultured cells; and comparing the measurementto a control value of the measured parameter, wherein the control valuecorresponds to a measurement of the same parameter for a cell cultureinto which said infectious pathogen has not been introduced.
 2. Themethod of claim 1, optionally comprising a culture matrix thatfacilitates growth of said host cells.
 3. The method of claim 1, whereinthe bioreactor is a rotating wall vessel.
 4. The method of claim 1,wherein said isolated host cells are epithelial cells.
 5. The method ofclaim 4, wherein said epithelial cells are human intestinal cells. 6.The method of claim 2, wherein said culture matrix consists ofmicrobeads or microcamers.
 7. The method of claim 1, wherein saidinfectious pathogen is selected from the group consisting of viruses,bacteria, protozoa, parasites and fungi.
 8. The method of claim 7,wherein said infectious pathogen is Salmonella typhimurium.
 9. Themethod of claim 1, wherein said culture medium comprises fetal bovineserum and a tri-sugar based medium comprising fructose, galactose andlactose.
 10. The method of claim 6, wherein said microbeads arecollagen-coated microbeads.
 11. A method of studying the infectivity ofa pathogen in tissues comprising the steps of: isolating cells from asample of intestinal epithelial tissue; placing said intestinalepithelial cells into a bioreactor comprising culture medium to form acell culture; applying sedimental shear stress to the cells in the cellculture to form a three dimensional tissue mass; seeding the formedtissue mass in a tissue culture vessel; introducing an infectiouspathogen to the formed tissue mass; assaying the infectivity of saidinfectious pathogen, wherein said assaying comprises the steps of:obtaining a measurement of at least one of the following parametersafter introducing said infectious pathogen: adherence of said infectiouspathogen to cultured cells; cytokine expression in cultured cells; orprostaglandin synthesis in cultured cells; and comparing the measurementto a control value of the measured parameter, wherein the control valuecorresponds to a measurement of the same parameter for a cell cultureinto which said infectious pathogen has not been introduced.
 12. Themethod of claim 11, wherein said infectious pathogen is Salmonellatyphimurium.
 13. A method of measuring the chemosensitivity of tissuesto a toxic material comprising: isolating cells from a host tissuesample; placing said isolated host cells into a bioreactor comprisingculture medium to form a cell culture; applying sedimental shear stressto the cells in the cell culture to form a three-dimensional tissuemass; seeding the formed tissue mass in a tissue culture vessel;introducing a toxic material into said three-dimensional tissue mass;and assaying the chemosensitivity of said toxic material by obtaining ameasurement of cell death among cultured cells after introduction ofsaid toxic material; and comparing the measurement to a control value ofcell death for a cell culture into which said toxic material has notbeen introduced.
 14. The method of claim 13, optionally comprising aculture matrix that facilitates growth of said host cells.
 15. Themethod of claim 13, wherein said isolated host cells are epithelialcells.
 16. The method of claim 15, wherein said epithelial cells arehuman renal cells.
 17. The method of claim 14, wherein said culturematrix consists of microbeads or microcarners.
 18. The method of claim17, wherein said microbeads are collagen-coated microbeads.
 19. Themethod of claim 13, wherein said toxic material is a chemotherapeuticmaterial.
 20. The method of claim 19, wherein said chemotherapeuticmaterial is an antibiotic.
 21. The method of claim 20, wherein saidantibiotic is gentamicin.
 22. The method of claim 13, wherein saidculture medium comprises fetal bovine serum and DMEM/F12.
 23. A methodof measuring the chemosensitivity of tissues to a toxic materialcomprising: isolating cells from a sample of human renal epithelialtissue; placing said isolated human renal epithelial cells into abioreactor comprising culture medium to form a cell culture; applyingsedimental shear stress to the cells in the cell culture to form athree-dimensional tissue mass; seeding the formed tissue mass in atissue culture vessel; contacting the three dimensional tissue mass witha toxic material; and assaying the chemosensitivity of said toxicmaterial by obtaining a measurement of cell death among cultured cellsafter introduction of said toxic material; and comparing the measurementto a control value of cell death for a cell culture into which saidtoxic material has not been introduced.
 24. The method of claim 23,wherein said toxic material is a chemotherapeutic material.
 25. Themethod of claim 24, wherein said chemotherapeutic material is anantibiotic.